5-nitro-2-furfural azines



United States Patent 3,296,257 S-NITRO-Z-FURFURAL AZINES James D. Johnston, Old Saybrook, Conn., assignor to Chas. Pfizer & Co., Inc., New York, N.Y., a corporation of Delaware No Drawing. Filed June 4, 1963, Ser. No. 285,195 Claims. (Cl. 260-440) This application is a continuation-in-part of Serial No. 803,376, filed April 1, 1959, now US. Patent No. 3,099,- 663, granted July 30, 1963.

This invention is concerned with antimicrobial agents and more particularly with a series of novel azine compounds which exhibit valuable activity against a variety of microorganisms.

I have made the discovery that compounds having the general formula in which A represents a carbonyl compound from which one carbonyl oxygen has has been removed, are remarkably effective antimicrobial agents showing activity against a variety of microorganisms and parasites, among them, organisms which cause disease in animals, including man, and fungi of industrial significance. In this formula A may take a variety of forms, but especially useful compounds are those in which A represents one of a particular group of substituents, as described hereinbelow.

A may, for example, be an alkanone from which the keto oxygen has been abstracted, as represented by the formula wherein R and R are alkyl groups, usually containing a total of from two to five carbon atoms. Higher alkyl groups may be introduced but ordinarily there will be no advantage. A may thus, for example, be =C(CH Also valuable are those compounds in which A represents cycloalkanone, usually of 5 to 7 carbon atoms, less the keto oxygen, for example /CH2CH2 A may likewise be alkanedione from which one keto oxygen has been removed. While a variety of alkanediones may be represented, those containing from 4 to 12 carbon atoms are preferred. A may thus, for example, represent such structures as CH3 CH3 CH3 CH3 l and l =CC=O =CCH2C=O In addition, A may advantageously be selected from keto-substituted alkanoic and alkanedioic acids, less the keto oxygen, or from formyl-substituted alkanoic acids, less the carboxaldehyde oxygen. Such substituents will preferably contain a total of up to 12 carbon atoms and will include, for example,

R1 :iuemnooon H0oo(CHz),o(oH2)zoooH and Rr-CH-COOH =6 Rr( HCOOH where R is hydrogen or alkyl, R is alkyl, and x and y are Zero or integers. In addition, the salts, such as the alkali-metal and amine salts, and esters, wherein the esterifying group is alkyl containing up to about 18 carbon atoms, may be employed in place of the corresponding acids.

Alternatively, A may be derived from an aldehyde by elimination of the carbonyl oxygen. Particularly suitable aldehydes include alkanal and alkenal containing up to 18 carbon atoms, as well as Z-furaldehyde, thiophene- 2-carboxaldehyde, monohydroxybenzaldehyde, and hemaldehydes having from one to five alkoxy substituents of from one to three carbon atom-s per alkoxy group. In addition, suitable aldehydes also include 1,4-dioxoquinoxaline carboxaldehyde and alkyl-substituted 1,4-dioxoquinoxaline carboxaldehydes in which the alkyl substituent contains from about one to five carbon atoms. They also include 'y-cyclohexenylcrotonaldehyde, a-rnethyl-'y-cyclohexenylcrotorraldehyde, and these crotonaldehydes with alkyl groups of from about one to five carbon atoms as ring substituents.

Among' the 1,4 dioxoquinoxaline carboxaldehydes from which the valuable new compounds may be derived are those of the formula wherein one of said R substituents, preferably in the hetero-cyclic ring, is forrnyl. The aromatic ring substituents may be hydrogen, alkyl, chloro, bromo, fluoro, hydroxy, lower lalkoxy, carboxy or lower carbalkoxy. Particular-ly preferred are those wherein one of the R and R substituents is formyl, the other being hydrogen or methyl, and wherein R R R and R are each selected from the group consisting of hydrogen and alkyl containing up to five carbon atoms.

The novel compounds of this invention exhibit in vitro and in vivo activity against a wide variety of microorganisms, including gram positive and gram negative bacteria. Eifectiveness is found, for example, against such organisms as Micrococcus pyogenes var. aureus, including antibiotic-resistant strains, Streptococcus pyogenes, Erysipelothrix rhusiopathiae, Corynebacterium diphtheriae, Bacillus subtilis, Clostridium: perfringens, Escherichia coli, Vibrio comma, Pasteurella multocida, Mycobacteriu-m 607, and Mycobacterium berolinense. Elfectiveness also is exhibited against a variety of other microorganisms, for example, protozoa such as Endomeba histolytica and Trichomonas vaginalis, and fungi such as Alternaria solani, Cladosporium cladosporoides, Trichophyton rubrum, Pythium debarynum, Aspergillus niger, and Penicillium funiculosum, as well as helminths, including Syphacia obvelata.

The compounds of this invention also exhibit activity against various species of Salmonella. Among these, a number of compounds are particularly elfective in the treatment of Salmonella infections in poultry. Each year a significant number of mortalities occur among poultry flocks, especially chickens, as a result of these infections, with a large economic loss resulting. The most important diseases of this nature in poultry result from infections by Salmonella gallinarum, which causes fowl tyfected with the organism by the oral route. One group serves as a control and receives no treatment. This group is fed a nutritionally adequate diet containing sufficient protein, carbohydrate, fat, vitamins and minerals to promote growth in healthy chicks. The other group receives this same basic diet except that the compound under test is admixed with the feed in suflicient quantity to provide a level of 0.1% by weight of active ingredient. The survival time of all chicks is recorded and from this data is calculated the ST that is the 50% survival time in days at the 95% confidence limit. The experiment is continued for seven days, at which time the surviving birds are sacrificed and the heart, spleen and liver recovered for quantitative determination of viable Salmonella organisms.

Compounds which are found to be particularly eflective in this test include, for example, 1-(5-nitro-2-furfural)-2-(dimethyl-fi-keto-glutarate) azine, 1-(5-nitro-2- furfura D-Z-(ethyl-3-ketobutyrate) azine, 1-(5-nitro-2-furfural)-24(acetone) azine, l-ethylpyruvate-2-(5-nitro-2- furfural) azine, 1-(5-nitro-2-furfural)-2-(Z-carboxaldehyde thiophene) azine, and 1-(5-nitro-2-furfural)-2=(dimethyl 3-keto-2-methyl-glutarate) azine. Especially valuable are I-(S-nitrofurfurylidene)-2-( 3,4 dimethoxy benzylidene) hydrazine, l-(5-nitrofurfurylidene)-2-(3 methyl-1,4-dioxo-2-quinoxalinylmethylene) hydrazine, l-(5 nitro-2- turfural)-2-(diethyl-B-ketoglutarate) azine, and 1-(5-nitr0- 2 furfural)-2-(dimethyl 2,4 dimethyl-3-ketoglutarate) azlne.

These new compounds are conveniently prepared by the condensation of S-nitrofurfural hydrazone with a carbonyl compound, in a solvent such as ethanol or aoetonitrile at refluxing temperature, according to the following general reaction where R and R are selected to conform to the structures hereinabove described. Alternatively, they may be prepared by condensing the hydrazone derived from the carbonyl compound with S-nitrofurfural, or its diacetate. Some of the carbonyl compounds are readily available, and the others may be synthesized by methods well known to those skilled in the art.

For anti-infective application, these new compounds may be blended with excipients or dispersed in diluents including water, isotonic saline, oils such as sesame oil, and the like. Many modes of administration are possible, including oral, subcutaneous, intramuscular, intravenous and topical application, the choice being dictated by the type and severity of the infection. For administration to poultry in the treatment of Salmonella infections, the compounds will ordinarily be administered orally, and may be admixed with feed to provide a concentration of at least about 0.001%, and preferably 0.01% or more by weight of active ingredient.

The following examples are given solely for the purpose of illustration and are not to be construed as limitations of this invention, many variations of which are possible without departing from the spirit or scope thereof.

Example I A mixture of 0.1 mole S-nitrofurfural hydrazone and 0.1 mole of dimethyl-2,4-dimethyl-3-keto-glutarate in 500 ml. acetonitn'le is heated at refluxing temperatures for about four hours. The resulting solution is filtered and the filtrate treated with activated carbon and evaporated in vacuum. The clear gum is treated with 250 ml. methanol and refrigerated. Filtration of the resulting crystal- 4. line slurry and recrystallization from methanol yields? 1- (5 nitro-2-furfural)-2(dimethyl-2,4-dimethyl-3-ketoglutarate) azine in the form of a crystalline solid melting at about 155 C.

Example II Following the procedure of Example I, diethyl acetonedicarboxylate is caused to react with S-nitrofurfural hydrazone, yielding 14.6 g. of 1-(5-nitro-2-furfural)-2-(diet-hyl-fl-ketoglutarate) azine in the form of orange plates melting at about 126 C. and having the following elemental analysis: carbon, 49.42%; hydrogen, 5.04%; nitrogen, 12.14%. Ultraviolet absorption maxima are observed at 3100 A. (E=15,100) and 4060 A. (e=29,25 0 in methanol). An infrared absorption maximum is observed at 5.755 microns;

Example III Following the procedure of Example I, the following series of compounds is prepared:

Rr- HC 0 0 R4 R3. R R4 CH1 H CH3 CH3 H Fl CH1 CH3 H Fl Cz'Fl-r C H7 H. 'H' CaHn C5H11 H- H CnHn 061113 11.... H CsHr-r 0811" H H CW 01 11 5 H IB W 15 31 H Fl' H C3117- Can-I CH3 CH3 CH1 CH-z Oqfl. H CH: 09H: N8 N8 CH3 CH3 NH3(C:H N H3(C2H5) Example IV According to the general procedure of Example I the following additional compounds are prepared from the corresponding carbonyl compounds and S-nitrofurfural where A represents one of the following carbonyl compounds with one carboxyl oxygen removed:

acetone methyl isobutyl ketone ethyl propyl ketone cyclopentanone cyclohexanone cycloheptanone 2,3-butanedione 2,4-pentanedione 4,9-dodecanedione 2-furaldehyde I 'y-(6,6-dimethyl-2-methylcyclohex-l-ene) crotonaldehyde a-methyI-q-cycIoheX-Z-ene crotonaldehyde a-methy1- -'(3-a-mylcyelohex-4-ene) crotonaldehyde a-methyl-y-(6,6-dimethyl-2-methylcyclohex-l-ene) crotonaldehyde glyoxylic acid ethyl 6-formyl hexanoate methyl -formyl decanoate ethyl pyruvate ethyl ,B-ketobutyrate butyl 'y-ketovalerate 1 l-keto-dodecanoic acid 8-keto-dodecanoic acid diethyl-a-ketomalon ate 4-keto-heptane-1,7-dioic acid -keto-undecane-hl l-dioic acid formaldehyde acetaldehyde isobutyraldehyde stearic aldehyde crotonaldehyde Example V The product of Example I is subjected to standard in vitro plate tests against a variety of microorganisms. The medium employed is prepared by adding 37 grams of dehydrated Bacto brain-heart infusion B37 (purchased from Difco Laboratories of Detroit) to a liter of distilled water and sterilizing the resulting solution in an autoclave. The compound under test is added to the brain-heart broth in various concentrations, up to 200 mcg. per ml., and the solutions are applied to agar plates seeded with one of the organisms. In this manner the minimum concentration of active ingredient necessary to inhibit organism growth for 24 hours at 37 C. is determined. Results are as follows:

Minimum inhibitory concentration, mcg./ml. Bacillus subtilis 25 Clostridium perfringens 25 Bacterium ammoniagenes 200 Aerobacter aerogenes 200 Proteus vulgaris 100 Pseudomonas aeruginosa 100 Erwinia amylovora 100 Desalfovibrio desulfuricalis 200 Vibrio comma 100 When S-nitrofurfural hydrazone is subjected to the same series of tests, it does not inhibit organism growth.

Example VI The products of Examples II, III and IV are subjected to similar screening tests and are found to be active against a wide variety of Gram-positive and- Gram-negative organisms.

Example VII The product of Example I is screened against Salmonella according to the same procedures, with the following results: a

Minimum inhibitory concentration, meg/ml.

S. typhosa 100 S. pullorum 25 S. gallinarum 50 Example VIII A typical poultry feed is prepared having the following compositions:

Delmix (commercially available mineral mix containing CaCO and small amounts of iron, zinc, manganese and other saltsLimestone Products Corporation of America, New Jersey) 0.1

Vitamin A (53-05 IU/lb.) 0.1 Vitamin D (681 ICU/lb.) 0.05 Klotogen F (commercially available form of vitamin K-Abbott Laboratories) 0.0003 Pyridoxine hydrochloride 0.0006 D,L-methionine 0. Niacin U.S.P. 0.0025 Choline chloride (25%) 0.2. Riboflavin 0.06 Calcium pantothenate (45%) 0.002 Myvamix (commercially available form of vitamin E) 0.05

The products of Examples I and II are added to different samples of this feed to provide compositions containing 0.1% by Weight of active ingredient. These compositions are successfully employed in the treatment of chicks infected with S. gallinarum, no toxic effects of the azine being observed. At the conclusion of the experiment the birds are sacrificed and the heart, spleen and liver found to be free of viable Salmonella.

When S-nitrofurfural hydrazone is employed in the same test, it is found to be toxic, causing weight loss and early death. At levels below 0.1%, it is inactive.

Example IX 2,3-dimethyl quinoxaline-1,4-dioxide, 20 g. in 250 ml. ethyl acetate is combined with 15 g. selenium dioxide. The reaction mixture is stirred under gentle reflux for 1- /2 hours and the ethyl acetate is then evaporated at reduced pressure. Selenium metal is then separated by filtration and the cake washed with methylene chloride. The combined filtrate and washings are then concentrated at reduced pressure to obtain 15.6 g. of 2-methyl-3-aldehydoquinoxaline-1,4-dioxide melting at 177179. Recrystallized from ethyl acetatemethylene chloride the purified substance is obtained in the form of prisms melting at 186-l87 (dec.). Analysis, calculated for C H O N z carbon 58.8%, hydrogen 3.95% nitrogen 13.72%; found: carbon 58.77%, hydrogen 3.93%, nitrogen 13.78%.

Example XI Following the procedure of the preceding example, the following aldehydo-quinoxaline-1,4-dioxides are prepared from the corresponding methylquinoxaline-1,4-dioxides.

a i s 1 R4 N u R3 R4 R5 R6 1 5 Formyl H H H H D0 H H n-Butyl..- n-Butyl... H D0 H Methyl Methyl- Methyl Methyl Do Methyldn H H H Methyl Formyl do H H Formyl--. Methyl H Methyl H H Methyl Formyl.-- H dn H H Formyl MethyL.-- H do Methyl H Do H Methy1 Ethyl H Dn H dn H Methyl Methyl PropyL..- H H H i-PropyL. H H H H H nAmyl H H Do Methyl Ethyl H H H Methy1 Formyl do Fr H H Those compounds which have a methyl substituent in the heterocyclic ring and which are unsymmetrically substituted in the aromatic ring are obtained as a mixture of the 2-formyl-3-methyl and the 2-methyl-3-formyl substances. These mixtures are separated by chromatography or by fractional crystallization. Suitable chromatographic adsorbents include alumina, silicic acid and Florisil (activated magnesium silicate). The material is applied to the column of adsorbent, developed and eluted in a solvent such as chloroform, benzene, methanol, or in a mixture of these. For fractional crystallization, appropriate solvent systems include ethyl acetate: methylene chlorideand acetonezhexane.

Example XII' 2-methyl-3-aldehydo-quinoxaline-1,4-dioxide, 5.1 g. (prepared as described hereinabove), is introduced to a warm solution of 3.9 g. S-nitrofurfural hydrazone in 250 ml. ethanol. The reaction mixture is heated at reflux temperature for 2 hours, with separation of red, crystalline material beginning after about 10 minutes reflux. The mixture is then refrigerated and 6.08 of red, crystalline 1 (S-nitrofurfurylidene)-2-(3methylrl,4-dioxo-2-quinoxalinylmethylene) hydrazine recovered by filtration. The product melts at 190-192". It exhibits in vitro activity against a variety of microorganisms, including bacteria, fungi and protozoa.

Mice infected with the rodent pinworm Syphacia obvelata are treated with the product once daily for two days by the oral route, and at the end of the second day the animals are sacrificed and the cecal content examined. The treated animals show a marked reduction in worms as compared with untreated controls, as follows:

Dosage level, mg./ kg. body wt. Percent efiiciency Example XIII 2 a i i OlN O GH=NN=HG R R4 \N/ B5 R4 R0 R1 Rs H H n H H Fl n-Buty n-Butyl- H Ff Methyl Methyl Methyl Methyl Methyl rln Fl Tl Do- H H H Methyl Dn Fr Methyl H H Do- H H Methyl H D0 Fl Methyl H H Methy Ethy H R dn Fl Methyl Methyl H Propyl H Fl H H i-Propyl- Fl Fl H H n-Amyl H H Methy Ethy H H H De. H H Ethyl These products exhibit activity against a variety of microorganisms and helminths.

Example XIV 1-(5-nitrofurfurylidene)-2(3,4-dimethoxy benzylidene hydrazine and I-(S-nitrofurfurylidene)-2-(3-methyl-1,4- dioxo-2-quinoxalinylmethylene) hydrazine are tested in vivo against S. gallinarum according to the procedure of 8 Example VIII and these compounds exhibitactivity at the 0.1% level.

Example XV The following compounds are tested against S. galli-.

narum according to the procedure of Example VIII and are found to exhibit activity:

wherein A is a substituted methylidene moiety derived from a carbonyl compound of the formula wherein one of said R and R substituents is formyl, the other of said R and R substituents is selected from the group consisting of hydrogen and methyl, and said R R R and R substituents are each selected from the group consisting of hydrogen and alkyl containing up to five carbon atoms.

2. A compound of the formula wherein A is a substituted methylidene moiety derived from a carbonyl compound selected from the group consisting of alkanone containing from 3 to 6 carbon atoms, cycloalkanone containing from 5 to 7 carbon atoms and alkanedione containing from 4 to 12 carbon atoms.

3. A compound of theformul-a wherein A is a substituted methylidene moiety derived from a carbonyl compound selected from the group consisting of alkanal containing up to 18 carbon atoms and alkenal containing up to 18 carbon atoms.

4. A compound of the formula HOCH I n H 02NC CH=NN=A sisting of monohydroxybenzaldehyde and alkoxybenzalclehyde containing from 1 to 3 carbon atoms in the alkoxy,

9 5. A compound of the formula HCCH O2N CH=NN=A wherein A is a substituted methylidene moiety derived from a carbonyl compound selected from the group consisting of y-cyciohexenylcrotonaldehyde, lower-alkyl-substituted -cyciohexenylcrotonaldehyde wherein the alkyl group is a ring substitnent, a-methyl-y-cycl-ohexenylcrotonaldehyde and lower-alkyl-substituted u-methyl-ycyclohexenylcrotonaldehyde wherein the alkyl group is a ring substituent.

References Cited by the Examiner UNITED STATES PATENTS 3,099,663 7/ 1963 Johnston 260-240 FOREIGN PATENTS 1,271,038 7/ 1961 France.

933,682 8/ 1963 Great Britain.

OTHER REFERENCES Israeli Patent 13,537, Dec. 22, 1960, abstracted in Chemical Abstracts, v01. 55, column 15509h (Aug. 7, 1961).

WALTER A. MODANCE, Primary Examiner.

H. I. MOATZ, Assistant Examiner. 

1. A COMPOUND OF THE FORMULA 